Lipoxygenase/hydroperoxide lyase generated compounds are thought to be important in plant defense. The effects of volatile compounds from this pathway on the proliferation of Escherichia coli TB1, Pseudomonas syringae pv. tabaci, and Pseudomonas syringae pv. angulata were evaluated. The vaporphase concentrations of compounds in the bioassay system were estimated by gas chromatography. At the highest concentrations tested, the C6 aldehyde (£)-2-hexenal completely inhibited proliferation of both P. syringae pathovars (570 /ig/L of air) and E. coli (930 jug/L of air). Similarly, the C6 alcohol (£)-2-hexen-l-ol prevented proliferation of P. syringae pathovars (1100 ng/L of air) and E. coli (2300 Mg/L of air). Among the bacteria tested, one isolate of P. syringae pv. angulata was the most sensitive to a lipoxygenase pathway volatile, exhibiting decreased proliferation after exposure to (£)-2-hexenal (40 Mg/L of air). The unsaturated volatiles, (£)-2-hexenal and (£)-2-hexen-l-ol, exhibited a greater inhibitory effect than the saturated volatiles, hexanal and 1-hexanol. The responses to the volatile compounds observed for £. coli TB1 and P. syringae pv. tabaci were similar and differed somewhat from that of P. syringae pv. angulata.

The sodium pump, Na,K-ATPase, is an important protein for maintaining intracellular ion concentration, cellular volume, and ion transport and is regulated both transcriptionally and post-transcriptionally. We previously demonstrated that hyperoxia increased Na,KATPase ␤ 1 gene expression in Madin-Darby canine kidney (MDCK) cells. In this study, we identify a DNA element necessary for up-regulation of the Na,K-ATPase ␤ 1 transcription by hyperoxia and evaluate the nuclear proteins responsible for this up-regulation. Transient transfection experiments in MDCK cells using sequential 5-deletions of the rat Na,K-ATPase ␤ 1 promoterluciferase fusion gene demonstrated promoter activation by hyperoxia between ؊102 and ؉151. The hyperoxia response was localized to a 7-base pair region between ؊62 and ؊55, which contained a GC-rich region consistent with a consensus sequence for the SP1 family, that was sufficient for up-regulation by hyperoxia. This GC element exhibited both basal and hyperoxia-induced promoter activity and bound both transcription factors SP1 and SP3 in electrophoretic mobility shift assays. In addition, electrophoretic mobility shift assays demonstrated increased binding of SP1/SP3 in cells exposed to hyperoxia while mutation of this element eliminated protein binding. Other GC sites within the proximal promoter also demonstrated up-regulation of transcription by hyperoxia, however, the site at ؊55 had higher affinity for SP proteins.

To assess the role of lipoxygenase (LOX; EC 1.13.11.12) in plants, we increased the expression of LOX in the tissues of Nicotiana tabacum L. cv. 'KY 14' by over-expression of the LOX2 gene from the soybean (Glycine max (L.) Merrill) embryo. The LOX2 cDNA was manipulated by replacing its 5'-untranslated sequence with the translational enhancer of the alfalfa mosaic virus (AMV), and subcloned into a plant expression vector, 3' to a duplicated cauliflower mosaic virus 35S promoter. The AMV-LOX2 construct was transferred into tobacco using Agrobacterium tumefaciens strain A281. The LOX2 was expressed in transgenic tobacco calli, leaves of transgenic plants, and their seed progeny at levels up to 0.1-0.2% of the total extracted protein. The introduced LOX2 affected fatty-acid oxidative metabolism as evidenced by a 50-529% increase in C6-aldehyde production. The impact on C6-aldehyde formation was greater than the effect on production of fatty-acid hydroperoxides. This is consistent with other studies indicating the greater propensity of soybean embryo LOX2 in generating C6-aldehydes than that of other well-characterized LOX isozymes.

When creating test cases for software, a common approach is to create tests that exercise requirements. Determining the adequacy of test cases, however, is generally done through inspection or indirectly by measuring structural coverage of an executable artifact (such as source code or a software model). We present ReqsCov, a tool to directly measure requirements coverage provided by test cases. ReqsCov allows users to measure Linear Temporal Logic requirements coverage using three increasingly rigorous requirements coverage metrics: naïve coverage, antecedent coverage, and Unique First Cause coverage. By measuring requirements coverage, users are given insight into the quality of test suites beyond what is available when solely using structural coverage metrics over an implementation.

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